The introduction of capillary electrophoresis (CE), capillary array electrophoresis (CAE), and subsequently microchip electrophoresis (μCE) has revolutionized biomedical research. For example, the completion of the Human Genome Project (HGP), two years ahead of schedule, was, in part, made possible by the development of automated, high-throughput capillary array electrophoresis DNA sequencing instruments. While impressive achievements have been made for the HGP, other de novo sequencing projects, for the comprehensive, comparative genetic analysis of humans as well as of agriculturally and industrially important plants, animals, insects, and microorganisms, will continue. Success of these efforts may well hinge on the development of microchip-based DNA sequencing systems that operate with replaceable polymer solutions for DNA separation—and the resulting easy automation and excellent reproducibility.
Size-dependent DNA separation is a most critical process in genome analysis. It is well-known that DNA mobility is size-independent in free-solution electrophoresis, because of the constant DNA charge-to-mass ratio. In a microchannel system (e.g., using fused silica capillaries of i.d. 50-100 μm), size-dependent electrophoretic DNA separation has been achieved, employing fluid, entangled polymer solutions such as water soluble cellulose derivatives polyethylene oxide copolymers or N-substituted acrylamide polymers. Usually, highly entangled solutions of high-molar-mass polymers are required for long DNA sequencing read lengths (the number of bases read without error in one single run). To date, the best sequencing performance has been obtained with ultra-high-molar-mass linear polyacrylamide (LPA) (molecular weights >10 million g/mole), prepared by inverse emulsion polymerization. Such a sieving network can produce up to 1000 bases in about 1 hour and 1300 bases in 2 hours under highly optimized CE and sample conditions (including optimized polymer molar mass distribution, matrix formulation, temperature, electric field, sample preparation and purification, injection, and base-calling algorithm), with routine performance of commercial LPA matrices at read lengths of about 600-800 bases.
Such LPA matrices, while highly effective for DNA separation, suffer from several drawbacks, in particular, high viscosity and lack of wall-coating ability. The extreme viscosity of high-molar-mass LPA solutions requires high pressure to initiate flow of the solution into microchannels. The necessity for high-pressure matrix replacement contributes significantly to the building and maintenance costs of microchannel electrophoresis instruments. Moreover, the low pressure tolerance of most plastic or glass chips (20-100 psi) prohibits such high-pressure loading for microfluidic devices. Another deficiency associated with LPA matrices is the need for wall modification to suppress electro-osmotic flow (EOF). Suppression of EOF promotes reproducible and efficient separations by eliminating wall-analyte interactions. Therefore, despite excellent DNA sieving performance, LPA does not fulfill criteria of an optimal sequencing matrix. Lower-viscosity, self-coating polymer matrices, such as poly-N,N-dimethylacrylamide (PDMA), polyethylene oxide (PEO) and polyvinylpyrrolidone (PVP), are available and have been used, but provide much shorter read lengths than LPA.
The development of polymeric matrices with “switchable viscosities” is one strategy to decouple the capillary loading and DNA separation properties. For example, “thermo-thinning” polymer networks undergo a thermodynamically driven volume-phase transition, accompanied by a dramatic decrease in viscosity, in response to a change in temperature over a narrow range. The temperature at which this phase transition occurs is termed the lower critical solution temperature (LCST) or the “cloud point” of the solution, and is characterized by a sharp increase in turbidity of the polymer solutions. Poly-N-isopropylacrylamide (pNIPA), with an LCST in water of 32° C., and hydroxypropylcellulose (HPC), with an LCST in water of 39° C., have been used as thermo-responsive sieving matrices for double-stranded (ds) DNA separations. Thermo-thinning polymer networks with designed LCSTs, based on linear copolymers of N,N-dimethylacrylamide (DMA) and N,N-diethylacrylamide (DEA), have been formulated as DNA sequencing matrices with a thermally controlled “viscosity switch.” In particular, a copolymer composed of 42% (w/w) DEA and 58% (w/w) DMA delivered 575 bases in 94 minutes with a base-calling accuracy of 98.5%. This copolymer network exhibits a dramatic drop in viscosity, of more than an order of magnitude, when heated above 80° C., which allows rapid matrix loading into the capillary lumen under very low applied pressure (50 psi). Upon reducing the temperature to below the LCST (to the sequencing temperature of 44° C.), the entangled state of the polymer coils in solution is restored as they redissolve in aqueous solution, providing effective DNA sequencing performance.
Another interesting class of polymer matrices shows “thermo-thickening” behavior: these polymer networks exhibit an upper critical solution temperature (UCST) at which an expansion of coil volume occurs, accompanied by thermo-association of polymer chains and a dramatic increase in viscosity. Thermo-gelation is thus actuated with an increase in temperature. An advantage of thermo-gelling networks is that they can be designed to allow microchannel loading at room temperature, and then heated to the sequencing temperature to gel. A number of thermo-thickening polymer matrices have been developed based on polymers that exhibit thermo-associative behavior, with novel co-polymer architectures such as poly-N-isopropylacrylamide-graft-polyethylene oxide (pNIPA-g-pEO), poly-N-isopropylacrylamide-graft-polyacrylamide (pNIPA-g-LPA), and polyethylene oxide-polypropylene oxide block copolymers (pEO-pPO-pEO). These polymers utilize the self-associating properties of the hydrophobic chain parts, which serve as physical crosslinking points, to form extended polymer networks when heated above the transition temperature. While it has been shown that these thermo-thickening polymer matrices can provide high-resolution dsDNA separations, single-base resolution of ssDNA under denaturing conditions (7 M urea, high temperature), as required for DNA sequencing, has not yet been presented in the literature for a thermo-gelling matrix.